Certain molecular events in eukaryotic cells depend on the existence or magnitude of an electric potential gradient across the plasma (i.e., outer) membrane of the cells. Among the more important of such events is the movement of ions across the plasma membrane through voltage-gated ion channels. Voltage-gated ion channels form transmembrane pores that open in response to changes in cell membrane potential and allow ions to pass through the membrane. Voltage-gated ion channels have many physiological roles. They have been shown to be involved in maintaining cell membrane potentials and controlling the repolarization of action potentials in many types of cells (Bennett et al., 1993, Cardiovascular Drugs & Therapy 7:195-202; Johnson et al., 1999, J. Gen. Physiol. 113:565-580; Bennett & Shin, “Biophysics of voltage-gated sodium channels,” in Cardiac Electrophysiology: From Cell to Bedside, 3rd edition, D. Zipes & J. Jalife, eds., 2000, W. B. Saunders Co., pp. 67-86; Bennett & Johnson, “Molecular physiology of cardiac ion channels,” Chapter 2 in Basic Cardiac Electrophysiology and Pharmacology, 1st edition, A. Zasa & M. Rosen, eds., 2000, Harwood Academic Press, pp. 29-57). Moreover, mutations in sodium, calcium, or potassium voltage-gated ion channel genes leading to defective channel proteins have been implicated in a variety of disorders including the congenital long QT syndromes, ataxia, migraine, muscle paralysis, deafness, seizures, and cardiac conduction diseases, to name a few (Bennett et al., 1995, Nature 376:683-685; Roden et al., 1995, J. Cardiovasc. Electrophysiol. 6:1023-1031; Kors et al., 1999, Curr. Opin. Neurol. 12:249-254; Lehmann et al., 1999, Physiol. Rev. 79:1317-1372; Holbauer & Heufelder, 1997, Eur. J. Endocrinol. 136:588-589; Naccarelli & Antzelevitch, 2000, Am. J. Med. 110:573-581).
Several types of voltage-gated ion channels exist. Voltage-gated potassium channels establish the resting membrane potential and modulate the frequency and duration of action potentials in neurons, muscle cells, and secretory cells. Following depolarization of the membrane potential, voltage-gated potassium channels open, allowing potassium efflux and thus membrane repolarization. This behavior has made voltage-gated potassium channels important targets for drug discovery in connection with a variety of diseases. Dysfunctional voltage-gated potassium channels have been implicated in a number of diseases and disorders. Wang et al., 1998, Science 282: 1890-1893 have shown that the voltage-gated potassium channels KCNQ2 and KCNQ3 form a heteromeric potassium ion channel known as the “M-channel.” Mutations in KCNQ2 and KCNQ3 in the M-channel are responsible for causing epilepsy (Biervert et al., 1998, Science 279:403-406; Singh et al., 1998, Nature Genet. 18:25-29; Schroeder et al., Nature 1998, 396:687-690).
Voltage-gated sodium channels are transmembrane proteins that are essential for the generation of action potentials in excitable cells (Catterall, 1993, Trends Neurosci. 16:500-506). In mammals, voltage-gated sodium channels consist of a macromolecular assembly of α and β subunits with the α subunit being the pore-forming component. α subunits are encoded by a large family of related genes, with some α subunits being present in the central nervous system (Noda et al., 1986, Nature 322:826-828; Auld et al., 1988, Neuron 1:449-461; Kayano et al., 1988, FEBS Lett. 228:187-194) and others in muscle (Rogart et al., 1989, Proc. Natl. Acad. Sci. USA 86:8170-8174; Trimmer et al., 1989, Neuron 3:33-49).
Voltage-gated calcium channels are transmembrane proteins that in the open configuration allow the passive flux of Ca2+ ions across the plasma membrane, down the electrochemical gradient. They mediate various cell functions, including excitation-contraction coupling, signal transduction, and neurotransmitter release.
Current methods of drug discovery often involve assessing the biological activity (i.e., screening) of tens or hundreds of thousands of compounds in order to identify a small number of those compounds having a desired activity. In many high throughput screening programs, it is desirable to test as many as 50,000 to 100,000 compounds per day. Unfortunately, current methods of assaying the activity of voltage-gated ion channels are ill suited to the needs of a high throughput screening program. Current methods often rely on electrophysiological techniques. Standard electrophysiological techniques involve “patching” or sealing against the cell membrane with a glass pipette followed by suction on the glass pipette, leading to rupture of the membrane patch (Hamill et al., 1981, Pflugers Arch. 391:85-100). This has limitations and disadvantages. Accessing the cell interior may alter the cell's response properties. The high precision optical apparatuses necessary for micromanipulating the cells and the pipettes make simultaneous recording from more than a few cells at a time impossible. Given these difficulties, the throughput that can be achieved with electrophysiological techniques falls far short of that necessary for high throughput screening.
Various techniques have been developed as alternatives to standard methods of electrophysiology. For example, radioactive flux assays have been used in which cells are loaded with a radioactive tracer (e.g., 86Rb+, 22Na+, [14C]-guanidinium) and the efflux of the dye is monitored. Cells loaded with the tracer are exposed to compounds and those compounds that either enhance or diminish the efflux of the tracer are identified as possible activators or inhibitors of ion channels in the cells' membranes.
Assays that measure the change in a cell's membrane potential due to the change in activity of an ion channel have been developed. Such assays often employ voltage sensitive dyes that redistribute between the extracellular environment and the cell's interior based upon a change in membrane potential and that have a different fluorescence spectrum depending on whether they are inside or outside the cell. A related assay method uses a pair of fluorescent dyes capable of fluorescence resonance energy transfer to sense changes in membrane potential. For a description of this technique, see González & Tsien, 1997, Chemistry & Biology 4:269-277. See also González & Tsien, 1995, Biophys. J. 69:1272-1280 and U.S. Pat. No. 5,661,035. Other methods employ ion selective indicators such as calcium dependent fluorescent dyes to monitor changes in Ca2+ influx during opening and closing of calcium channels.
Ideally, methods of screening against voltage-gated ion channels require that the transmembrane potential of the cells being assayed be controlled and/or that the ion channels studied be cycled between open and closed states. This has been done in various ways. In standard electrophysiological techniques, the experimental set-up allows for direct manipulation of membrane potential by the voltage clamp method (Hodgkin & Huxley, 1952, J. Physiol. (Lond.) 153:449-544), e.g., changing the applied voltage or injecting various ions into the cell. In other methods, changing the extracellular K+ concentration from a low value (e.g., 5 mM) to a higher value (e.g., 70-80 mM) results in a change in the electrochemical potential for K+ due to the change in the relative proportion of intracellular and extracellular potassium. This results in a change in the transmembrane electrical potential towards a more depolarized state. This depolarization can activate many voltage-gated ion channels, e.g., voltage-gated calcium, sodium, or potassium channels. Alternatively, Na+ channels can be induced into an open conformation by the use of toxins such as veratridine or scorpion venom (Strichartz et al., 1987, Ann. Rev. Neurosci. 10:237-267; Narahashi & Harman, 1992, Meth. Enzymol. 207:620-643). While sometimes effective, such experimental manipulations may alter the channel pharmacology, can be awkward to perform, and can lead to artifactual disturbances in the system being studied.
Electrical field stimulation of cells has been performed on a single cell by sealing a glass microelectrode to the cell membrane. Rupture of the sealed patch of cell membrane resulted in an electrical connection between the interior fluid in the glass microelectrode and the fluid within the cell that was used to stimulate the cell via an electronic pulse generator. The electrophysiological response of the cell was measured via a sensitive electronic amplifier. The disadvantage of this technique is that only one cell at a time was tested and it is a tedious and time consuming operation to seal the microelectrode to an individual cell.
HEK293 cells have been grown on a silicon chip made up of an array of field-effect transistors. Some of the cells were positioned over the gate region of the transistors, thus having portions of their plasma membranes overlying the source and the drain. When a patch pipette in such cells manipulated the intracellular voltage, Maxi-K potassium channels in the cells' plasma membranes were opened. This led to current flow in the region between the cells' membrane and the transistor. This current flow modulated the source-drain current, which could be detected by an appropriate device. The chip plus cells was said to have potential as a sensor and as a prototype for neuroprosthetic devices. See Straub et al., 2001, Nature Biotechnol. 19:121-124; Neher, 2001, Nature Biotechnol. 19:114.